Treatment and composition for wound healing

ABSTRACT

A method and medicament for promoting wound healing in a subject is disclosed. The medicament comprises an effective amount of an agent comprising one or more of; (i) an activated protein C (APC), (ii) a functional fragment of an APC, (iii) an APC mimetic compound, and (iv) protein C. Delivery systems including gels, sponges, gauzes and meshes incorporating the agent for topical administration are also described.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of medicine and, moreparticularly, to wound healing and methods and compositions forpromoting wound healing. In a particular application of the presentinvention, activated protein C (APC) is administered to a slow healingwound by, for example, topical application.

BACKGROUND TO THE INVENTION

[0002] Wounds are internal or external bodily injuries or lesions causedby physical means, such as mechanical, chemical, viral, bacterial, orthermal means, which disrupt the normal continuity of structures. Suchbodily injuries include contusions, wounds in which the skin isunbroken, incisions, wounds in which the skin is broken by a cuttinginstrument, lacerations, and wounds in which the skin is broken by adull or blunt instrument. Wounds may be caused by accidents or bysurgical procedures.

[0003] The healing of wounds is a complex process involving a number ofstages. These include; 1) coagulation, which begins immediately afterinjury; 2) inflammation, which begins a few minutes later; 3) amigratory and proliferative process (granulation stage), which beginswithin hours to days; and 4) a remodelling process with subsequentdevelopment of full strength skin (1-3).

[0004] Coagulation and Inflammation

[0005] Coagulation controls haemostasis and initiates healing byreleasing a variety of growth factors and cytokines from degranulatedplatelets. During the inflammation phase, platelet aggregation anddotting form a matrix which traps plasma proteins and blood cells toinduce the influx of various types of cells. Neutrophils are the firstcells to arrive and function to phagocytise contaminating bacteria,digest the fibrin clot and release mediators to attract macrophages andactivate fibroblasts and keratinocytes (3). Macrophages digestpathogens, debride the wound and secrete cytokines/growth factors (eginterleukin-1(IL-1), epidermal growth factor (EGF), vascular endothelialgrowth factor (VEGF), transforming growth factor-β(TGF-β), and basicfibroblast growth factor (bFGF)) that stimulate fibroblasts andendothelial cells. Overall, the inflammatory stage is important to guardagainst infection and promote the migratory and proliferative stages ofwound healing.

[0006] Granulation and Remodeline of the Extracellular Matrix

[0007] These stages include cellular migration and proliferation.Although lymphocytes and macrophages are involved, the predominant celltypes are epithelial, fibroblast and endothelial. Within hours of aninjury, an epidermal covering, comprised mainly of keratinocytes, beginsto migrate and cover the epidermis, a process known asre-epithelialisation. When they completely cover the wound theydifferentiate and stratify to form a new epidermis with a basal lamnina.Angiogenesis (ie the formation of new blood vessels) occurs during thisstage and provides nutrients for the developing tissue to survive.Fibroblasts migrate into the wound site and produce collagen andproteoglycans which ultimately give the wound tensile strength. As theremodelling phase progresses, granulation tissue is replaced by anetwork of collagen and elastin fibers leading to the formation of scartissue.

[0008] Failed Wound Healing

[0009] Impaired dermal wound healing and/or dermal ulcers occur inpatients with peripheral arterial occlusive disease, deep veinthrombosis, diabetes, pressure sores and burns (4). Despite intenseinvestigation, the molecular mechanisms associated with impaired woundhealing are poorly understood.

[0010] Wound healing is affected by numerous factors, including localfactors (eg growth factors, edema, ischemia, infection, arterialinsufficiency, venous insufficiency or neuropathy), systemic factors (eginadequate perfusion and metabolic disease) and other miscellaneousfactors, such as nutritional state, exposure to radiation therapy andsmoking.

[0011] Leucocytes, particularly neutrophils, and macrophages persist inthe surrounding tissue and secrete a range of proteases, includingmatrix metalloproteinases (MMPs) and serine proteases (5). Excessiveaccumulation of these enzymes interferes with the matrix remodelling(6). It is thought that agents which inhibit proteases will benefitwound healing (7). Another feature of some chronic wounds is thereduction or absence of angiogenesis, which prevents nutrients fromaccessing the newly formed tissue (8).

[0012] Existing Technologies to Improve Wound Healing

[0013] Chronic wounds are initially managed by treatment comprisingeschar debridement, antibiotic treatment where appropriate, and regulardressing (2). Other dressings, such as hydrogels, hydrocolloids, oralginates, may also be used. Venous ulceration is treated by compressiontherapy, whereas arterial or diabetic ulcers require regular changes ofdressings. Pressure sores are encouraged to heal by the relief ofpressure at the injury site. Some other physical devices such as lasertreatment, hyperbaric oxygen and electrical stimulation for arterialulcers, are also used to promote wound healing (2, 9, 10).

[0014] For wounds that are unresponsive to such interventions, the useof tissue-engineered skin, such as Dermagraft or Apligraf, is an option.This therapy acts to prevent bacterial infection and allows the woundthe chance to heal by normal reparative processes (11, 12). The use ofsuch skin replacements to accelerate wound healing depends on theavailability of an existing vascular supply in the existing wound.

[0015] Another approach to wound healing involves the administration ofgrowth factors/cytokines, which have been shown to accelerate cellproliferation in vitro and/or to promote wound healing in some animalmodels. These include IL-1, platelet-derived growth factor (PDGF), EGF,VEGF, TGF-β, and bFGF (2). Procuren (Curative Technologies), anautologous platelet releasate, contains at least five growth factors,that aid in the formation of granulation tissue andre-epithelialisation. This autologous growth factor mnix has achievedsome success in human subjects with ulcerated limb lesions (13).However, on the whole, results from most clinical trials using growthfactors/cytokines have been disappointing. For example, EGF failed toheal venous stasis ulcers and IL-1 failed to treat pressure soreseffectively (2). Similar results were reported using bFGF (14). Thereason for the lack of efficacy is not certain, but may relate to themultifactorial effects, some undesirable for healing, of growthfactors/cytokines.

[0016] Thus, there is an ongoing need to identify and develop new agentsfor the promotion of wound healing.

[0017] Activated protein C (APC) is a serine protease having a molecularweight of about 56 kD that plays a central role in physiologicalanticoagulation. The inactive precursor, protein C, is a vitaminK-dependent glycoprotein synthesised by the liver and endothelium and isfound in plasma. Activation of protein C occurs on the endothelial cellsurface and is triggered by a complex formed between thrombin andthrombomodulin (15,16). Another endothelial specific membrane protein,endothelial protein C receptor (EPCR), has been shown to accelerate thisreaction more than 1000-fold (17). Endothelial APC functions as ananticoagulant by binding to the co-factor, protein S, on the endothelialsurface, which inactivates the clotting factors Factor VIIIa and FactorVa. The importance of APC as an anticoagulant is reflected by thefindings that deficiencies in this molecule result in familial disordersof thrombosis (18).

[0018] Recently, it has also been reported that APC additionally acts asan anti-inflammatory agent and directly activates the protease,gelatinase A (17, 20). Gelatinase A is secreted by many different celltypes, including smooth muscle cells, fibroblasts and endothelial cells.By degrading the coilagens present in the basement membrane (21) andallowing cells to invade the stroma, gelatinase A plays an importantrole in physiological remodelling and angiogenesis (22). Gelatinase Aalso plays an important role in numerous diseases, such as promoting theinvasion of thymic epithelial tumors (23), promoting the destruction ofthe joint in arthritis by cleaving collagen from the cartilage matrix(24) and contributing to cardiac mechanical dysfunction duringreperfusion after ischemia (25). In addition to its ability to degradethe matrix, gelatinase A can also target other substrates. For example,it cleaves big endothelin-1 to yield a potent vasoconstrictor,implicating gelatinase A as a regulator of vascular reactivity (26).Gelatinase A release can also mediate platelet aggregation (27).

[0019] Further, and as is demonstrated in the examples providedhereinafter, APC is also able to promote regeneration of endothelialcells after wounding in vitro, stimulate re-epithelialisation fibroblastinvasion and angiogenesis in a chicken embryo and enhance wound healingin a rat wounding model. These functions when taken together with theabovementioned anticoagulating, anti-inflammatory and GelatinaseA-activating functions, strongly indicate that APC, functional fragmentsthereof, and the precursor of APC (ie protein C) is/are useful for thetreatment of wounds and, particularly, slow-healing wounds.

SUMMARY OF THE INVENTION

[0020] Thus, in a first aspect, the present invention provides a methodfor promoting wound healing in a subject, said method comprisingadministering to said subject an effective amount of an agent comprisingone or more of;

[0021] (i) an APC,

[0022] (ii) a functional fragment of an APC,

[0023] (iii) an APC mimetic compound, and

[0024] (iv) protein C,

[0025] optionally in admixture with a pharmaceutically-acceptablecarrier.

[0026] In a second aspect, the present invention provides a medicamentfor promoting wound healing in a subject, said medicament comprising anamount of an agent comprising one or more of;

[0027] (i) an APC,

[0028] (ii) a functional fragment of an APC,

[0029] (iii) an APC mimetic compound, and

[0030] (iv) protein C,

[0031] in admixture with a pharmaceutically-acceptable carrier.

[0032] In a third aspect, the present invention provides a deliverysystem (eg a gel, sponge, gauze or mesh) incorporating an amount of anagent comprising one or more of;

[0033] (i) an APC,

[0034] (ii) a functional fragment of an APC,

[0035] (iii) an APC mimetic compound, and

[0036] (iv) protein C,

[0037] said delivery system being suitable for application to a woundand thereafter promoting wound healing.

[0038] In a fourth aspect, the present invention provides the use of anagent comprising one or more of;

[0039] (i) an APC,

[0040] (ii) a functional fragment of an APC,

[0041] (iii) an APC mimetic compound, and

[0042]0 (iv) protein C,

[0043] for the preparation of a medicament for promoting wound healingin a subject.

[0044] In a fifth aspect, the present invention provides the use of anagent comprising one or more of;

[0045] (i) an APC,

[0046] (ii) a functional fragment of an APC,

[0047] (iii) an APC mimetic compound, and

[0048] (iv) protein C,

[0049] for the preparation of a delivery system, said delivery systembeing suitable for application to a wound and thereafter promoting woundhealing.

DETAILED DISCLOSURE OF THE INVENTION

[0050] The APC and/or protein C utilised in the present invention may beobtained by purification from a suitable source (eg blood taken fromhumans or other animals) or produced by standard recombinant DNAtechniques such as is described in, for example, Maniatis, T. et al.,Molecular Cloning: a laboratory manual, Second Edition, Cold SpringHarbor Laboratory Press. Recombinant APC or protein C may incorporatemodifications (eg amino acid substitutions, deletions, and additions ofheterologous amino add sequences), which may, for example, enhancebiological activity or expression of the respective protein. However,preferably, the present invention utilises human APC and/or protein C.The APC and/or protein C may also be glycosylated by methods well knownin the art and which may comprise enzymatic and non-enzymatic means.

[0051] Suitable functional fragments of an APC may be produced bycleaving purified natural APC or recombinant APC with well knownproteases such as trypsin and the like, or more preferably, byrecombinant DNA techniques or peptide/polypeptide synthesis. Suchfunctional fragments may be identified by generating candidate fragmentsand assessing biological activity by, for example, assaying foractivation of MMP-2, promotion of repair of a wounded endothelialmonolayer and/or angiogenesis in chicken embryo chorio-alantoic membrane(CAM) in a manner similar to that described in the examples providedherein. Preferably, functional fragments will be of 5 to 100 amino acidsin length, more preferably, of 10 to 30 amino acids in length. Thefunctional fragments may be linear or circularised and may includemodifications of the amino add sequence of the native APC sequence fromwhence they are derived (eg amino acid substitutions, deletions, andadditions of heterologous amino acid sequences). The functionalfragments may also be glycosylated by methods well known in the art andwhich may comprise enzymatic and non-enzymatic means.

[0052] Suitable APC mimetic compounds (ie compounds which mimic thefunction of APC) may be designed using any of the methods well known inthe art for designing mimetics of peptides based upon peptide sequencesin the absence of secondary and tertiary structural information (28).For example, peptide mimetic compounds may be produced by modifyingamino acid side chains to increase the hydrophobicity of defined regionsof the peptide (eg substituting hydrogens with methyl groups on aromaticresidues of the peptides), substituting amino acid side chains withnon-amino acid side chains (eg substituting aromatic residues of thepeptides with other aryl groups), and substituting amino- and/orcarboxy-termini with various substituents (eg substituting aliphaticgroups to increase hydrophobicity). Alternatively, the mimetic compoundsmay be so-called peptoids (ie non-peptides) which include modificationof the peptide backbone (ie by introducing amide bond surrogates by, forexample, replacing the nitrogen atoms in the backbone with carbonatoms), or include N-substituted glycine residues, one or more D-aminoacids (in place of L-amino acid(s)) and/or one or more α-amino acids (inplace of β-amino acids or γ-amino acids). Further mimetic compoundalternatives include “retro-inverso peptides” where the peptide bondsare reversed and D-amino acids assembled in reverse order to the orderof the L-amino acids in the peptide sequence upon which they are based,and other non-peptide frameworks such as steroids, saccharides,benzazepine1,3,4-trisubstituted pyrrolidinone, pyridones andpyridopyrazines. Suitable mimetic compounds may also bedesigned/identified by structural modelling/determnination, by screeningof natural products, the production of phage display libraries (29),minimised proteins (30), SELEX (Aptamer) selection (31), combinatoriallibraries and focussed combinatorial libraries, virtualscreening/database searching (32), and rational drug design techniqueswell known in the art (33).

[0053] The present invention is suitable for promoting wound healinggenerally, but is particularly suitable for application to the promotionof slow-healing wounds, otherwise known as “chronic wounds”,“impaired-healing wounds” or “ulcers”, and may be of any of the woundtypes discussed above. However, preferably, the present invention isapplied to wounds selected from the group consisting of;

[0054] (i) dermal ulcers such as those associated with pressure,vasculitis, arterial and venous diseases (eg in patients suffering fromdiabetes, in aged patients, associated with venous insufficiency andcerebrovascular incidents, and resulting from pressure sores orlocalised areas of tissue damage resulting from direct pressure on theskin or from shearing forces and friction),

[0055] (ii) burns,

[0056] (iii) oral wounds (eg caused by gingivitis),

[0057] (iv) eye wounds (eg corneal wounds resulting from injury, surgeryor laser therapy),

[0058] (v) non-cutaneous wounds (eg stomach/oesophageal ulcers, vaginalulcers and internal injury or surgery (including plastic surgery),

[0059] (vi) ischemia-reperfusion injury (eg resulting from myocardialinfarction),

[0060] (vii) bone and cartilage damage as occurs in musculoskeletaldisorders such as rheumatoid arthritis and osteoarthritis, and

[0061] (viii) warfarin-related skin necrosis.

[0062] The agent may be one or a mixture of any or all of the groupconsisting of an APC, a functional fragment of an APC, an APC mimeticcompound, and a protein C.

[0063] The agent is preferably administered to a subject after asufficient period of time since wounding has elapsed such that thecoagulation/inflammation stages of the wound healing process havesubstantially concluded. In practice, it is preferable thatadministration of the agent occur within 1 to 48 hours after wounding,more preferably within 1 to 1 hours after wounding.

[0064] The agent may be administered to a subject through oral orsystemic routes or by direct application (e.g. topical administration)to the wound as a medicament formulation, or as incorporated into adelivery system (eg gelatin sponge such as Gelfoam, fine gauze, nylonmesh, or an adhesive plastic strips such as a Band-aid™) which isapplied to the wound.

[0065] The effective amount of the agent may be expected to varydepending upon the type, site and seriousness of the wound to betreated. It would be well within the skill of persons skilled in the artto adjust the amount appropriately to obtain optimal results. It is,however, expected that generally the effective amount of the agent willbe in the range of 0.01 to 10000 μg per kg of body weight, morepreferably between 0.1 and 1000 μg per kg of body weight, and mostpreferably between about 1 and 200 μg per kg of body weight.

[0066] Medicaments according to the present invention preferably includean amount of the agent in the range of 0.01 to 1000 μg per g ofmedicament, in admixture with a pharmaceutically-acceptable carrier (eggelatin and/or collagen for cream or gel medicaments; isotonic saline, aphosphate buffered solution or the like for drops; or materials such asstarch, gelatin, agar, sugar, carboxymethylcellulose, polyvinylalcohol,magnesium stearate, and sodiumalginate for dry powders). For use withnon-cutaneous wounds and ischemnia-reperfusion injury, the medicamentsof the present invention may be formulated for oral or systemicadministration.

[0067] Medicaments and delivery systems (ie gels, sponges, gauzes andmeshes) according to the present invention, may contain one or moreother active compounds or substances such as other molecules involved inthe protein C pathway (eg protein S, EPCR, factor V/Va or factorVIII/VIIIa); antimicrobial agents such as chlorhexidine, povidine-iodineand ciprofloxacin; anticoagulants such as heparin or antithrombin III;steroids such as dexamethasone; inhibitors of inflammation;cardiovascular drugs such as calcium channel blockers; cytokines/growthfactors such as epidermal growth factor; local anaesthetics such asbupivacaine; antitumor drugs such as taxol; polyclonal, monoclonal orchimeric antibodies, or functional derivatives or fragments thereof suchas antibodies to regulate cell proliferation.

[0068] Further, where the medicaments and delivery systems according tothe present invention utilise protein C, the medicaments and deliverysystems may also include a suitable amount of an agent for activatingthe protein C (eg thrombin, kallikrein and/or thrombomodulin).

[0069] Throughout this specification the word “comprise”, or variationssuch as “comprises” or “comprising”, will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

[0070] Any discussion of documents, acts, materials, devices, articlesor the like which has been included in the present specification issolely for the purpose of providing a context for the present invention.It is not to be taken as an admission that any or all of these mattersform part of the prior art base or were common general knowledge in thefield relevant to the present invention as it existed in Australia orelsewhere before the priority date of each claim of this application.

[0071] The invention will hereinafter be further described by way of thefollowing non-limiting examples and accompanying figures.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

[0072]FIG. 1 shows the effect of APC on repair of a “wounded” foreskinendothelial (FSE) cell monolayer in culture. Cell monolayers werescraped with a pipette tip, washed and covered with medium containingvarious amounts of APC, PMA or no treatment. The cells were incubated at37 degrees C. for 24 hr before being measured for wound closure asdescribed in the example. Results shown represent the number of cellsmigrating into the wound (mean±SD) per selected unit of area ofduplicate wounds. Similar results were obtained in two separateexperiments.

[0073]FIG. 2 shows the effect of APC on chicken embryo chorio-allantoicmembrane (CAM). Chicken embryos were cracked into a sterile weigh boaton day 1. On day 4, gelatin sponges were treated with 5 μg APC inphosphate buffered saline (PBS) or PBS alone and then placed on top ofthe CAM. On day 9, the embryos were fixed in Bouin's fluid, sectionedperpendicular to the top surface of the CAM, stained with Masson's stainand viewed under light microscopy. APC-treated CAM showing completere-epithelialisation over sponge (thin arrows) (A) and many new bloodvessels (thick arrows) and fibroblast infiltration (hatched arrows) (B).In the PBS-treated sponges there is no epithelial regrowth (thickarrows) and negligible invasion of cells into the sponge (C,D).

[0074]FIG. 3 shows the effect of APC on a rat wound healing model.Sprague-Dawley rats were wounded and then treated with A) saline or B)20 μg APC and photographed after 4 days. C) Summary of results using 2rats (total 8 wounds) each treated with saline or 20 μg APC. Resultsshown are mean +/−S.D of wound area (mm²) after 40 hr, 4 days and 7days.

[0075]FIG. 4 shows the effect of different doses APC on a rat woundhealing model. Normal rats were wounded using an 8 mm punch biopsy andimmediately treated with 0 μg (Control, 3 rats, 12 wounds), 10 μg (3rats, 12 wounds), 40 μg (4 rats, 16 wounds), 70 μg (3 rats, 12 wounds)or 100 μk (3 rats, 12 wounds) of APC. Wound size was measured by imageanalysis after 1, 3, 5, 7 and 9 days and results shown are mean +/−S.Dof wound area (mm²) after 1, 2, 3, 4 , 5, 7 and 9 days.

[0076]FIG. 5 shows the effect of APC on a rat wound healing model.Normal rats were wounded using an 8 mm punch biopsy and not treated(Control, 2 rats, 7 wounds) or treated twice with APC (40 μg APCimmediately after wounding followed by a second treatment of 40 μg APCafter 1 day) (3 rats, 11 wounds). Wound size was measured by imageanalysis and results shown are mean +/−S.D of wound area (mm²) after 1,2, 3, 4 , 5, 7 and 9 days

[0077]FIG. 6 shows the effect of APC on a diabetic rat wound healingmodel. Streptozotocin-induced diabetic rats were wounded using an 8 mmpunch biopsy and immediately treated with 20 μg APC (2 rats, 7 wounds)or no test agent (Control; 1 rat, 4 wounds). Wound closure was assessedvisually and after 1, 2, 3, 4, 5, 7 and 9 days. There was a significantdifference in the rate of wound healing (slope of the regression lines)between the control and APC-treated rats, with the latter healing faster(p<0.01).

EXAMPLE 1

[0078] APC Promotion of Endothelial Wound Repair

[0079] Activated protein C (APC) was tested for its ability to promoterepair of endothelial wounding using a modification of an in vitroassay, as described previously (19). Briefly, confluent microvascularendothelial cells (FSE) from neonatal foreskins were cultured for 5 daysin 24-well culture plates in growth medium (Biorich plus 50 μg/mlheparin, 50 μg/ml endothelial cell growth supplement and 5% humanserum). The endothelial monolayers were wounded by a single strokeacross the diameter of the well with a pipette tip. The media anddislodged cells were then aspirated, and the plates rinsed with Hanksbuffer. Fresh growth medium was added to the plates along with APC atvarious concentrations or the potent tumour-promoting angiogenic factor,phorbol myristate acetate (PMA) (10 ng/ml) and the cells were incubatedat 37 degrees C. After 24 hr, the width of the wound was visualisedmicroscopically and results at different doses of APC were quantifiedusing image analysis and a dose-response curve was generated (FIG. 1).Cells cultured in the presence of 75 nM APC achieved almost completewound closure within 24 hours, and showed more than twice the migratoryresponse than did cells without APC. At 100 nM there was no furtherincrease. APC (75 nM) had approximately the same activity as PMA.

EXAMPLE 2

[0080] APC Promotion of Angiogenesis

[0081] In view of the ability of APC to activate gelatinase A andpromote endothelial wounding, APC was investigated as to whether itcould promote angiogenesis. APC was added to the chicken embryochorio-allantoic membrane (CAM) assay using gelatin sponges (Gelfoam).Sponges were cut to approximately 2 mm×2 mm. Five μg APC in phosphatebuffered saline (PBS) or PBS alone was added to gelatin sponges whichwere subsequently placed on the 9 day old CAM, as previously described(34). The CAMs were inspected daily and on day 14 were photographed andfixed for histological sectioning. Macroscopically, on day 14, theAPC-treated gelatin sponges were surrounded by blood vessels that grewradially inwards towards the sponge in a “spoke-wheel” pattern (data notshown). In contrast, gelatin sponges treated with PBS had no surroundingvascular formation. Histological sections showed that APC-treatedsponges were infiltrated with many new blood vessels (angiogenesis)(FIG. 2). In addition, there was a large migration of fibroblasts intothe APC-treated sponges. There was also marked proliferation of theepithelial layer, with the ectoderm completely growing over the gelatinsponge. Associated with this re-epithelialisation was stratification andinvolution at the periphery of the sponge. The endoderm alsodemonstrated stratification with villous formation in some sections andthe presence of cells being shed from the villi (not shown). In contrastto the APC-treated sponges, there was little evidence of anyre-epithelialisation, endothelial cell or fibroblast infiltration in thePBS control sponges.

EXAMPLE 3

[0082] APC Promotion of Wound Healing

[0083] In view of APC's ability to stimulate endothelial migration andenhance re-epithelialisation, fibroblast invasion and angiogenesis, APCwas examined for a capacity to improve wound healing in a rat model.Sprague-Dawley rats were anaesthetised and four full-thickness woundswere excised, using a 8 mm punch biopsy, on the back of the rat,exposing the underlying dorsolateral skeletal muscle fascia. Hemostasiswas achieved by even compression with sterile gauze. APC was diluted inisotonic, sterile, pyrogen-free saline solution and each excision wastreated with a 50 μl topical application of sterile, pyrogen-free salinesolution or saline containing 20 μg APC. The wounds were left open withno dressing and rats caged one per cage. Wound closure was assessedvisually and after 40 hr, 4 days and 7 days. At each timepoint thewounds were digitally photographed using a Nikon Coolpix 950, with adistance calibration scale in the frame. The area of the wound wascalculated by image analysis (Scion Image). After 40 hr there was amarked visual improvement in the wound closure in APC-treated woundscompared to controls. On day 4 image analysis results revealed asignificant reduction in wound size of APC-treated wounds compared tocontrols (FIG. 3). This difference was maintained on day 7 (FIG. 3c,**p<0.01, ***p<0.001).

EXAMPLE 4

[0084] APC Promotion of Wound Healing

[0085] APC was further examined for a capacity to improve wound healingin a rat model. Sprague-Dawley rats were anaesthetised and fourfull-thickness wounds were excised, using a 8 mm punch biopsy, on theback of the rat, exposing the underlying dorsolateral skeletal musclefascia. Hemostasis was achieved by even compression with sterile gauze.APC was diluted in isotonic, sterile, pyrogen-free saline solution andeach excision was immediately treated with a 50 μl topical applicationof sterile, pyrogen-free saline solution or saline containing thefollowing: 0 g APC (Control, 3 rats, 12 wounds), 10 g APC (3 rats, 12wounds), 40 μg APC (4 rats, 16 wounds ), 70 μg APC (3 rats, 12 wounds)or 100 μg APC (3 rats, 12 wounds). The wounds were left open with nodressing and rats caged one per cage. Wound size was measured by imageanalysis after 1, 3, 5, 7 and 9 days. At each timepoint, the wounds weredigitally photographed using a Nikon Coolpix 995. The area of the woundwas calculated by image analysis (Scion Image). Results are shown inFIG. 4. After 1 day, there was a significant reduction in the size ofthe wounds treated with 10 or 40 μg APC. There was no difference betweenthe controls and rats treated with 70 or 100 μg APC. The significantreduction in wound size was most notable with 40 μg APC and seen on days1, 3, 7 and 9.(**p<0.01, * p<0.05, Student's t-test, using CoStat).

EXAMPLE 5

[0086] APC Promotion of Wound Healing

[0087] APC was further examined for a capacity to improve wound healingin a rat model. Sprague-Dawley rats were wounded as described in Example4. APC was diluted in isotonic, sterile, pyrogen-free saline solutionand each excision was treated with a 50 μl topical application ofsterile, pyrogen-free saline solution or saline containing 40 μg APC.After 48 hr, wounds were treated with a second application of 40 μg APC.The wounds were left open with no dressing and rats caged one per cage.Wound closure was assessed visually and after 1, 2, 3, 4, 5, 7 and 9days. At each timepoint, the wounds were digitally photographed using aNikon Coolpix 995. The area of the wound was calculated by imageanalysis (Scion Image). Results are shown in FIG. 5. There was asignificant difference in the size of the wounds after 1 day, withAPC-treated rats healing faster than controls (p<0.01). This differencewas also observed at days 2, 3 and 7 (**p<0.01, *p<0.05, Studentzz'st-test, using CoStat).

EXAMPLE 6

[0088] APC Promotion of Wound Healing in Diabetic Rats

[0089] APC was examined for a capacity to improve wound healing in adiabetic rat model. The diabetic model was selected because it is awell-described model for slow wound healing (35). Diabetes was inducedin Sprague-Dawley rats using the standard procedure of an IP injectionof streptozotocin. After 1 week the blood glucose levels of the ratswere >20 mM, indicative of diabetes. Diabetic rats were wounded using an8 mm punch biopsy, as described above in Example 4, and immediatelytreated with 20 μg APC (2 rats, 7 wounds) or no test agent (Control, 1rat, 4 wounds). The wounds were left open with no dressing and ratscaged one per cage. Wound closure was assessed visually and after 1, 2,3, 4, 5, 7 and 9 days. At each timepoint, the wounds were digitallyphotographed using a Nikon Coolpix 995. The area of the wound wascalculated by image analysis (Scion Image). Results are shown in FIG. 6.There was a significant difference in the rate of wound healing (slopeof the regression lines) between the control and APC-treated rats, withthe latter healing faster (p<0.01).

[0090] Conclusion

[0091] The ability of APC to repair endothelial wounding promotere-epithelialisation, fibroblast infiltration and angiogenesis, as wellas accelerate wound healing in the rat, indicates that it will be aneffective wound healing agent.

[0092] References

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[0128] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

1. A method for promoting wound healing in a subject, said methodcomprising administering to said subject an effective amount of an agentcomprising one or more of; (i) an activated protein C (APC), (ii) afunctional fragment of an APC, (iii) an APC mimetic compound, and (iv)protein C, optionally in admixture with a pharraceutically-acceptablecarrier.
 2. The method of claim 1, wherein the agent is APC.
 3. Themethod of claim 2, wherein the agent is human APC.
 4. The method ofclaim 1, wherein the agent is protein C.
 5. The method of claim 4,wherein the agent is human protein C.
 6. The method of any one of claims1, 4 and 5, wherein the protein C is administered with an activator forprotein C.
 7. The method of claim 6, wherein the activator is selectedfrom the group consisting of thrombin, kalikrein and/or thrombomodulin.8. The method of claim 1, wherein the agent is a functional fragment ofAPC.
 9. The method of claim 1, wherein the agent is an APC mimeticcompound.
 10. The method of any one of claims 1 to 9, wherein the agentis administered to said subject within 1 to 10 hours of wounding. 11.The method of any one of claims 1 to 10, wherein the wound for whichwound healing is to be promoted is selected from the group consisting ofdermal ulcers, burns, oral wounds, eye wounds, non-cutaneous wounds,ischemia-reperfusion injury, bone and cartilage damage andwarfarin-related skin necrosis.
 12. The method of claim 11, wherein thewound for which wound healing is to be promoted is a dermal ulcer. 13.The method of any one of claims 1 to 12, wherein the agent isadministered topically.
 14. The method of any one of claims 1 to 13,wherein the effective amount of the agent is in the range of 0.1 to1000μg per kg of body weight.
 15. The method of claim 14, wherein theeffective amount of the agent is in the range of 0.1 to 10 μg per kg ofbody weight.
 16. A medicament for promoting wound healing in a subject,said medicament comprising an amount of an agent comprising one or moreof; (i) an activated protein C (APC), (ii) a functional fragment of anAPC, (iii) an APC mimetic compound, and (iv) protein C, in admixturewith a pharmaceutically-acceptable carrier.
 17. The medicament of claim16, wherein the agent is human APC.
 18. The medicament of claim 16,wherein agent is human protein C.
 19. The medicament of claim 16 or 18,further comprising an activator for the protein C.
 20. The medicament ofclaim 19, wherein the activator is selected from the group consisting ofthrombin, kallikrein and/or thrombomodulin.
 21. The medicament of anyone of claims 16 to 20, wherein the medicament is for treatment of awound selected from the group consisting of dermal ulcers, burns, oralwounds, eye wounds, non-cutaneous wounds, ischemia-reperfusion injury,bone and cartilage damage and warfarin-related skin necrosis.
 22. Themedicament of claim 21, wherein the medicament is for treatment of adermal ulcer.
 23. The medicament of any one of claims 16 to 22, whereinthe medicament is for topical administration.
 24. A delivery systemincorporating an amount of an agent comprising one or more of; (i) anactivated protein C (APC), (ii) a functional fragment of an APC, (iii)an APC mimetic compound, and (iv) protein C, said delivery system beingsuitable for application to a wound and thereafter promoting woundhealing.
 25. The delivery system of claim 24, wherein the agent is humanAPC.
 26. The delivery system of claim 24, wherein the agent is humanprotein C.
 27. The delivery system of claim 24 or 26, further comprisingan activator for the protein C.
 28. The delivery system of claim 27,wherein the activator is selected from the group consisting of thrombin,kallikrein and/or thrombomodulin.
 29. The delivery system of any one ofclaims 24 to 28, wherein the delivery system is a gel, sponge, gauze ormesh.
 30. The use of an agent comprising one or more of; (i) anactivated protein C (APC), (ii) a functional fragment of an APC, (iii)an APC mimetic compound, and (iv) protein C, for the preparation of amedicament for promoting wound healing in a subject.
 31. The use ofclaim 30, wherein the agent is human APC.
 32. The use of claim 30,wherein the agent is human protein C.
 33. The use of claim 32, whereinthe medicament further comprises an activator for the protein C.
 34. Theuse of claim 33, wherein the activator is selected from the groupconsisting of thrombin, kallikrein and/or thrombomodulin.
 35. The use ofany one of claims 30 to 34, wherein the medicament is for treatment of awound selected from the group consisting of dermal ulcers, burns, oralwounds, eye wounds, non-cutaneous wounds, ischemia-reperfusion injury,bone and cartilage damage and warfarin-related skin necrosis.
 36. Theuse of claim 35, wherein the medicament is for treatment of a dermalulcer.
 37. The use of any one of claims 30 to 36, wherein the medicamentis for topical administration.
 38. The use of an agent comprising one ormore of; (i) an activated protein C (APC), (ii) a functional fragment ofan APC, (iii) an APC mimetic compound, and (iv) protein C, for thepreparation of a delivery system, said delivery system being suitablefor application to a wound and thereafter promoting wound healing. 39.The use of claim 38, wherein the agent is human APC.
 40. The use ofclaim 38, wherein the agent is human protein C.
 41. The use of claim 40,wherein the delivery system further comprises an activator for theprotein C.
 42. The use of claim 40, wherein the activator is selectedfrom the group consisting of thrombin, kallikrein and/or thrombomodulin.43. The use of any one of claims 38 to 42, wherein the delivery systemis a gel, sponge, gauze or mesh.